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Drosophila retinal degeneration C (RDGC) is the founding member of the PPEF family of protein phosphatases. RDGC mediates dephosphorylation of the visual pigment rhodopsin and the TRP ion channel. From the rdgC locus, three protein isoforms, termed RDGC-S, -M, and -L, with different N-termini are generated. Due to fatty acylation, RDGC-M and -L are attached to the plasma membrane while RDGC-S is soluble. To assign physiological roles to these RDGC isoforms, we constructed flies that express various combinations of RDGC protein isoforms. Expression of the RDGC-L isoform alone did not fully prevent rhodopsin hyperphosphorylation and resulted in impaired photoreceptor physiology and in decelerated TRP dephosphorylation at Ser936. However, expression of RDGC-L alone as well as RDGC-S/M was sufficient to prevent degeneration of photoreceptor cells which is a hallmark of the rdgC null mutant. Membrane-attached RDGC-M displayed higher activity of TRP dephosphorylation than the soluble isoform RDGC-S. Taken together, in vivo activities of RDGC splice variants are controlled by their N-termini.

Untreated periodontal disease may influence general health. However, how may a physician, who is not trained in periodontal probing, detect untreated periodontitis? Activated matrix metalloproteinase-8 (aMMP-8) in saliva correlates with periodontal probing parameters. Thus, sensitivity and specificity of a chair-side test for aMMP-8 to detect periodontitis were evaluated. Thirty cases [untreated chronic periodontitis (ChP); 15 generalized moderate and 15 generalized severe] and 30 controls [probing depths (PD) ≤3 mm, vertical probing attachment level (PAL-V) ≤2 mm at

Alkylglycerol monooxygenase (AGMO; EC 1.14.16.5) is the only enzyme known to cleave the O-alkyl ether bond of alkylglycerols in humans. It is an integral membrane protein with nine predicted transmembrane domains. We attempted to express and purify full-length and truncated forms of AGMO in Escherichia coli. Full-length AGMO could not be expressed in three different E. coli expression strains, three different expression vectors and several induction systems. We succeeded, however, in expression of three N-terminally strep-tagged truncated forms, named active sites 1, 2 and 3, with 205, 134 and 61 amino acids, respectively. Active site 1 fragment, containing two predicted transmembrane regions, a membrane associated region and all known amino acid residues important for catalytic activity, was not fully soluble even in 8 M urea. Active site 2 containing only one predicted membrane associated domain required 8 M urea for solubilisation and eluted in gel filtration in 1 M urea as a trimer. Active site 3 with no hydrophobic domain eluted in gel filtration in 1 M urea as monomer and dimer. These results show that even truncated forms of AGMO are barely soluble when expressed in E. coli and show a high tendency for aggregation.

In the past decade, the World Wide Web has been subject to dramatic changes. Web sites have evolved from static information resources to dynamic and interactive applications that are used for a broad scope of activities on a daily basis. To examine the consequences of these changes on user behavior, we conducted a long-term client-side Web usage study with twenty-five participants. This report presents results of this study and compares the user behavior with previous long-term browser usage studies, which range in age from seven to thirteen years. Based on the empirical data and the interview results, various implications for the interface design of browsers and Web sites are discussed. A major finding is the decreasing prominence of backtracking in Web navigation. This can largely be attributed to the increasing importance of dynamic, service-oriented Web sites. Users do not navigate on these sites searching for information, but rather interact with an online application to complete certain tasks. Furthermore, the usage of multiple windows and tabs has partly replaced back button usage, posing new challenges for user orientation and backtracking. We found that Web browsing is a rapid activity even for pages with substantial content, which calls for page designs that allow for cursory reading. Click maps provide additional information on how users interact with the Web on page level. Finally, substantial differences were observed between users, and characteristic usage patterns for different types of Web sites emphasize the need for more adaptive and customizable Web browsers.

The quantification of pharmacological parameters at G protein-coupled receptors (GPCRs) is indispensable in drug research but costly and time-consuming when conventional methods are sequentially applied. With neuropeptide Y (NPY) Y(1), Y(2), and Y(5) receptors as model systems, a homogenous flow cytometric method for the simultaneous determination of the affinity, selectivity, and activity of GPCR ligands was developed. Mixtures of cells expressing the receptors of interest and cyanine-labeled NPY as a universal fluorescent Y(1), Y(2), and Y(5) receptor agonist were used. Calcium mobilization was measured in different channels with the aid of fluo-4 and fura red. A combination of dye-loaded HEL-Y(1) and CHO-Y(2)-Galpha(qi5) cells with unloaded HEC-1B-Y(5) cells allowed the simultaneous determination of Y(1), Y(2), and Y(5) receptor selectivity preceded by the Y(1) and Y(2) receptor-mediated response with one and the same sample. The data are in good agreement with those determined by radioligand binding and spectrofluorimetry. The convenient, robust, and inexpensive multiparametric procedure offers a broad range of applications in the pharmacological characterization of GPCR ligands.

As part of a programme to develop fluorescence-based methods for the study of the interactions between G-protein coupled receptors (GPCRs) and their ligands the preparation of low molecular weight fluorescence-labelled neuropeptide Y (NPY) Y(5) antagonists is reported. The naphthylsulfonyl group in the potent quinazoline-type NPY Y(5) receptor antagonist CGP 71683A was replaced with a dansyl, nitrobenzoxadiazole (NBD) or acridine-9-carbonyl group. In radioligand binding studies on human Y(5) receptor expressing HEC-1B cells the substances labelled with acridine (K(i) 311 nM) and NBD (K(i) > 1000 nM) proved to be moderately active or inactive, respectively. By contrast, a K(i) value of 49 nM was found for the dansyl analogue compared to 2 nM for CGP 71683A. No binding to Y(1) receptors (SK-N-MC cells, displacement of [(3)H]propionyl-NPY) was detected for the new compounds at concentrations

The aim of the study is to compare detection frequency of periodontal pathogens in patients with aggressive/severe chronic periodontitis using pooled plaque samples from the deepest pockets per quadrant/per sextant. In 100 patients with aggressive/chronic periodontitis, subgingival plaque was sampled from the deepest pockets per quadrant (MT4) and per sextant (MT6). Plaque samples were taken using two sterile paper points simultaneously. One paper point from each pocket was pooled with the three other paper points of the pockets (MT4). Subsequently, the remaining four paper points were pooled with two paper points from the deepest pockets from the two remaining sextants (MT6). The content of each vial was analyzed with nucleic-acid based methods for Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, Treponema denticola, Prevotella intermedia, Parvimonas micra, Fusobacterium nucleatum, Campylobacter rectus, Eubacterium nodatum, Eikenella corrodens, and Capnocytophaga sp. The detection frequency of A. actinomycetemcomitans (MT4/MT6) at 22/24 %, T. forsythia at 93/96 %, P. gingivalis at 78/79 %, T. denticola at 88/90 %, P. intermedia at 40/46 %, P. micra at 75/79 %, F. nucleatum at both 99 %, C. rectus at 84/89 %, E. nodatum at 62/65 %, E. corrodens at 80/87 %, and Capnocytophaga sp. at 49/58 % was higher with MT6 than with MT4. None of these differences were statistically significant. The detection frequency of the investigated periopathogens was statistically insignificant higher with the sampling method MT6 compared with MT4. In daily dental practice, the plaque sampling of the deepest pockets per quadrant seems to be sufficient.